Journal: International Journal of Molecular Medicine

Article Title: RUNX3 modulates hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells

doi: 10.3892/ijmm.2017.2998

Figure Lengend Snippet: RT-PCR primer sequences.

Article Snippet: Rabbit anti-RUNX3 (bs-0378R) , Bioss , 5% non-fat milk , 1:1,000.

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: RUNX3 modulates hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells

doi: 10.3892/ijmm.2017.2998

Figure Lengend Snippet: Primary antibodies.

Article Snippet: Rabbit anti-RUNX3 (bs-0378R) , Bioss , 5% non-fat milk , 1:1,000.

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: RUNX3 modulates hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells

doi: 10.3892/ijmm.2017.2998

Figure Lengend Snippet: Western blotting of runt-related transcription factor 3 (RUNX3) and its targeting proteins in the control, hypoxia, RUNX3i and green fluoresent protein (GFP) groups.

Article Snippet: Rabbit anti-RUNX3 (bs-0378R) , Bioss , 5% non-fat milk , 1:1,000.

Techniques: Western Blot

Journal: International Journal of Molecular Medicine

Article Title: RUNX3 modulates hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells

doi: 10.3892/ijmm.2017.2998

Figure Lengend Snippet: Hypoxia enhances runt-related transcription factor 3 (RUNX3) expression in human cardiac microvascular endothelial cells (HCMECs). (A) RT-PCR data showing the mRNA level of RUNX3 in the control, hypoxia, RUNX3i and green fluoresent protein (GFP) groups. Results were normalized to that of reference gene β-actin (bars indicate mean ± SD, n=7; * P<0.05 vs. control group; # P<0.05 vs. hypoxia group; ▲ P<0.05 vs. RUNX3i group). (B and C) Relative RUNX3 and hypoxia inducible factor-1α (HIF-1α) protein expression to GAPDH in the HCMECs from each group (n=7, * P<0.05 vs. control group; # P<0.05 vs. hypoxia group; ▲ P<0.05 vs. RUNX3i group).

Article Snippet: Rabbit anti-RUNX3 (bs-0378R) , Bioss , 5% non-fat milk , 1:1,000.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: RUNX3 modulates hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells

doi: 10.3892/ijmm.2017.2998

Figure Lengend Snippet: Knockdown of runt-related transcription factor 3 (RUNX3) attenuates endothelial-mesenchymal transition (EndMT) of human cardiac microvascular endothelial cells (HCMECs). (A) RT-PCR data showing the mRNA expression levels of the endothelial markers (CD31 and VE-cadherin) and mesenchymal markers [fibroblast-specific protein (FSP)-1 and α-smooth muscle actin (α-SMA)] in the HCMECs. Results were normalized to reference gene β-actin (n=7, * P<0.05 vs. control group; # P<0.05 vs. hypoxia group; ▲ P<0.05 vs. RUNX3i group). (B) Relative protein expression of CD31, VE-cadherin, FSP-1 and α-SMA to GAPDH in the HCMECs from each group (n=7, * P<0.05 vs. control group; # P<0.05 vs. hypoxia group; ▲ P<0.05 vs. RUNX3i group). (C) Double immunofluorescence staining with antibodies to CD31 (red) and α-SMA (green). Nuclei were counterstained with DAPI (blue). The expression of CD31 was downregulated and α-SMA upregulated in the hypoxia group compared with the control group. Scale bars, 50 μ m. (D) Double immunofluorescence staining with antibodies to CD31 (red) and α-SMA (blue). The expression of CD31 was upregulated and α-SMA was downregulated in the RUNX3i group compared with the green fluoresent protein (GFP) group. Scale bars, 50 μ m.

Article Snippet: Rabbit anti-RUNX3 (bs-0378R) , Bioss , 5% non-fat milk , 1:1,000.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Double Immunofluorescence Staining

Journal: International Journal of Molecular Medicine

Article Title: RUNX3 modulates hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells

doi: 10.3892/ijmm.2017.2998

Figure Lengend Snippet: Low expression of runt-related transcription factor 3 (RUNX3) induces angiogenesis and decreases the migration of human cardiac microvascular endothelial cells (HCMECs). (A and B) Tube formation and Transwell migration assays (Scale bars, 50 μ m). (C and D) Tube formation counts and the amounts of Transwell cells (n=7, * P<0.05 vs. control group; # P<0.05 vs. hypoxia group; ▲ P<0.05 vs. RUNX3i group). (E) Western blotting of vascular endothelial growth factor receptor 1 (VEGFR1) in the HCMECs from each group. (F) Relative protein expression of VEGFR1 to GAPDH in the HCMECs from each group (n=7, * P<0.05 vs. control group; # P<0.05 vs. hypoxia group; ▲ P<0.05 vs. RUNX3i group).

Article Snippet: Rabbit anti-RUNX3 (bs-0378R) , Bioss , 5% non-fat milk , 1:1,000.

Techniques: Expressing, Migration, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: RUNX3 modulates hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells

doi: 10.3892/ijmm.2017.2998

Figure Lengend Snippet: Runt-related transcription factor 3 (RUNX3) regulates expression of principal endothelial-mesenchymal transition (EndMT) transcriptional factors. (A) Gene expression of Snail and Slug by RT-PCR. Results were normalized to that of reference gene β-actin (n=7, * P<0.05 vs. control group; # P<0.05 vs. hypoxia group; ▲ P<0.05 vs. RUNX3i group). (B and C) Relative protein expression of Snail and Slug to GAPDH in the human cardiac microvascular endothelial cells (HCMECs) from each group (n=7, * P<0.05 vs. control group; # P<0.05 vs. hypoxia group; ▲ P<0.05 vs. RUNX3i group).

Article Snippet: Rabbit anti-RUNX3 (bs-0378R) , Bioss , 5% non-fat milk , 1:1,000.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: RUNX3 modulates hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells

doi: 10.3892/ijmm.2017.2998

Figure Lengend Snippet: Crosstalk of hypoxia-induced endothelial-mesenchymal transition (EndMT) signaling pathways. Under hypoxic conditions, transcription factor hypoxia inducible factor-1α (HIF-1α) protein is accumulated and functionally activated, which induces the expression of genes associated with EndMT, such as Notch1, transforming growth factor-β (TGF-β), and runt-related transcription factor 3 (RUNX3), and the stabilization of Snail and Slug. RUNX3 is likely to be a direct target of HIF-1α, which can stabilize the Notch-ICD. The Notch-ICD also promotes the expression of EndMT-associated genes. Hypoxia can also activate the TGF-β signaling pathway through Smad2/3 proteins to induce EndMT transcription factor expression. ICD, intracellular domain.

Article Snippet: Rabbit anti-RUNX3 (bs-0378R) , Bioss , 5% non-fat milk , 1:1,000.

Techniques: Expressing

Journal: The Journal of comparative neurology

Article Title: Comparative analyses of the Smith-Magenis syndrome protein RAI1 in mice and common marmoset monkeys.

doi: 10.1002/cne.25589

Figure Lengend Snippet: FIGURE 5 RAI1 and TCF20 are highly colocalized and form an evolutionarily conserved protein complex in the cortex. (a) Immunofluorescence staining shows that RAI1 and TCF20 are highly colocalized throughout all layers in the adult marmoset prefrontal cortex. Scale bars: 20 μm. (b) Immunofluorescence staining shows that RAI1 and TCF20 are highly colocalized throughout all layers in the adult mouse prefrontal cortex. Scale bars: 20 μm. (c) Quantification shows that more than 70% of TCF20+ cells express RAI1 (top) and that more than 70% of RAI1+ cells express TCF20 (bottom). (d) Top panel: immunoprecipitation using an anti-RAI1 antibody identifies RAI1–TCF20 interaction in cortical extracts of newborn Rai1-FLAG mouse. Bottom panel: western blotting using an anti-FLAG antibody confirms the successful pull-down of RAI1. (e) Immunoprecipitation experiments using an anti-TCF20 antibody show that TCF20 interacts with RAI1-FLAG (top panel), PHF14 (middle panel), and HMG20A (bottom panel) in cortical extracts of newborn Rai1-FLAG mice. Western blotting using an anti-TCF20 antibody confirms a successful pull-down of TCF20. (f) Immunoprecipitation experiments using an anti-TCF20 antibody show that TCF20 interacts with RAI1-FLAG (top panel), PHF14 (top panel), and HMG20A (bottom panel) in cortical extracts from adult Rai1-FLAG mice. Western blotting using an anti-TCF20 antibody confirms a successful pull-down of TCF20. (g) Immunoprecipitation experiments using an anti-TCF20 antibody show that TCF20 interacts with RAI1 (top panel), PHF14 (middle panel), and HMH20A (bottom panel) in cortical extracts of newborn marmosets. Western blotting using an anti-TCF20 antibody confirms a successful pull-down of TCF20. (h) Immunoprecipitation experiments using an anti-TCF20 antibody show that TCF20 interacts with RAI1 (top panel), PHF14, and HMG20A (bottom panels) in cortical extracts of adult marmosets. Western blotting using an anti-TCF20 antibody confirms a successful pull-down of TCF20. The positive bands of the immunoprecipitation reactions are marked with red asterisks and the input bands are marked with green asterisks (*). All immunoprecipitation experiments were performed two to three times (also see Figure S4). Note that some proteins showed increased molecular weight after IP enrichment, which is not uncommon in IP experiments. A potential explanation includes the enrichment of post-translationally modified proteins.

Article Snippet: Antibody Antigen Host and type Dilution Source, #cat, RRID CaMKII Full-length CamKII, clone 6G9 Mousemonoclonal IgG 1:200 Abcam ab22609 AB_447192 CD31 CD31/PECAM-1 (immunogenmouse Glu18-Lys590) Goat polyclonal IgG 1:20 (marmoset)1:50 (mouse) R&D systems AF3628AB_2161028 FLAG DYKDDDDK, cloneM2 Mousemonoclonal IgG 1:1000 (WB) Sigma F1804 AB_262044 GAD67 Recombinant GAD67 protein, clone 1G10.2 Mousemonoclonal IgG 1:500 MilliporeMAB5406 AB_2278725 GAPDH GAPDH fusion protein Ag0766, clone 1E6D9 Mousemonoclonal IgG 1:1000 (WB) ProteinTech 60004-1-Ig AB_2107436 GFAP Recombinant full-length humanGFAP Chicken polyclonal IgY 1:1000 Abcam ab4674 AB_304558 HMG20A HumanHMG20A amino acids 1-347 Rabbit polyclonal IgG 1:1000 (WB) Thermo-Fisher 12085-2-AP AB_2117587 MEIS2 HumanMEIS2 aa1-381, clone 1H4 Mousemonoclonal IgG 1:200 Abnova H00004212-M01 AB_425545 NEUN Purified cell nuclei frommouse brain, clone A60 Mousemonoclonal IgG 1:500 MilliporeMAB377 AB_2298772 NEUROD2 HumanNeuroD2 amino acids 23-37 Goat polyclonal IgG 1:500 St John’s Lab STJ72554 AB_2927398 NR1 Fusion protein containing sequence from the intracellular loop between transmembrane regions III and IV of NR1, clone 54.1 Mousemonoclonal IgG 1:100 Thermo-Fisher 32-0500 AB_2533060 PHF14 Human PHF14, 16 amino acids from theN-terminal half Rabbit polyclonal IgG 1:1000 (WB) Millipore ABE1359-25UL AB_2924888 Phospho-Histone H3 Phospho-HistoneH3 (Ser10), clone 6G3 Mousemonoclonal IgG 1:100 Cell Signaling Technology 9706 AB_331748 RAI1 Mouse Rai1 amino acids 1738-1756 Rabbit polyclonal IgG 1:200 (IF) In-houseAB_2921229 RAI1 Human RAI1 amino acids 1-100 Rabbit polyclonal IgG 1:1000 (WB) AbcamAB_1925378 RARα Recombinant Human RARα, clone 9α−9A6 Mousemonoclonal IgG 1:500 Millipore 04-1545 AB_10615821 REELIN Mouse Reelin amino acids 150-500, clone G10 Mousemonoclonal IgG 1:500 Abcam ab78540 AB_1603148 S100β Bovine brain S-100β, clone SH-B1 Mousemonoclonal IgG 1:500 Sigma S2532 AB_477499 SOX2 SRY (sex determining region Y)-box 2 Rabbit polyclonal IgG 1:100 Thermo-Fisher PA1-094AB_2539862 TBR1 T-box braintranscription factor 1 Chicken polyclonal antibodies 1:100 Millipore AB2261AB_10615497 TCF20 Human TCF20 amino acids 1909-1958 Rabbit polyclonal IgG 1:100 (IF) Millipore SAB2106444 AB_2921230 GFAP Recombinant Protein corresponding to aa1 to 432 from humanGFAP Polyclonalguinea pig antiserum 1:1000 Synaptic Systems 173004 residing near the cortical ventricles, and we found coexpression of GFAPwith a proliferating cellular marker p-HH3.

Techniques: Immunofluorescence, Staining, Immunoprecipitation, Western Blot, Molecular Weight, Modification

Journal: The Journal of comparative neurology

Article Title: Comparative analyses of the Smith-Magenis syndrome protein RAI1 in mice and common marmoset monkeys.

doi: 10.1002/cne.25589

Figure Lengend Snippet: FIGURE 6 Human TCF20 bi-directionally regulates human RAI1 protein abundance. (a) Illustrations show two independent pairs of sgRNAs used to induce genetic deletion of human RAI1. (b) Four independent RAI1-knockout (KO) clones of human 293A cells were generated using two pairs of sgRNAs (guide-pairs). KO1 and KO2 were generated by guide-pair 1, while KO3 and KO4 were generated by guide-pair 2. Representative western blotting data show that RAI1 protein levels decrease in KO clones 1–4. By contrast, TCF20, PHF14, and HMG20A protein levels remain unchanged. (c) Quantification shows that in four different RAI1-KO clones, the protein levels of RAI1 (but not the other three RAI1-interacting proteins) decrease. N = 6 independent replications for each clone. ns: not significantly different, one-way ANOVA. (d) Illustration shows a pair of sgRNAs that induce TCF20 deletion in human cells. (e) Western blotting assays show that endogenous TCF20 protein levels significantly decrease in two independent TCF20-KO clones, confirming successful TCF20 knockout. RAI1 protein levels decrease, while PHF14 and HMG20A protein levels remain unchanged. (f) Quantification shows that in two independent TCF20-KO clones, the protein levels of RAI1 (but not PHF14 or HMG20A) decrease. N = 3 independent replications for each clone. *p < .05, one-way ANOVA. (g) Quantitative RT-PCR shows that RAI1 mRNA levels are slightly upregulated in TCF20-KO clones. *p < .05, one-way ANOVA. (h) Left: western blotting data show that TCF20 overexpression increases RAI1 protein abundance. Right: quantification shows significantly increased RAI1 protein levels upon TCF20 overexpression. Three independent biological replicates, *p < .05, unpaired t-test. (i) Western blotting shows that overexpressing TCF20 increases RAI1 protein levels but not PHF14 or HMG20A.

Article Snippet: Antibody Antigen Host and type Dilution Source, #cat, RRID CaMKII Full-length CamKII, clone 6G9 Mousemonoclonal IgG 1:200 Abcam ab22609 AB_447192 CD31 CD31/PECAM-1 (immunogenmouse Glu18-Lys590) Goat polyclonal IgG 1:20 (marmoset)1:50 (mouse) R&D systems AF3628AB_2161028 FLAG DYKDDDDK, cloneM2 Mousemonoclonal IgG 1:1000 (WB) Sigma F1804 AB_262044 GAD67 Recombinant GAD67 protein, clone 1G10.2 Mousemonoclonal IgG 1:500 MilliporeMAB5406 AB_2278725 GAPDH GAPDH fusion protein Ag0766, clone 1E6D9 Mousemonoclonal IgG 1:1000 (WB) ProteinTech 60004-1-Ig AB_2107436 GFAP Recombinant full-length humanGFAP Chicken polyclonal IgY 1:1000 Abcam ab4674 AB_304558 HMG20A HumanHMG20A amino acids 1-347 Rabbit polyclonal IgG 1:1000 (WB) Thermo-Fisher 12085-2-AP AB_2117587 MEIS2 HumanMEIS2 aa1-381, clone 1H4 Mousemonoclonal IgG 1:200 Abnova H00004212-M01 AB_425545 NEUN Purified cell nuclei frommouse brain, clone A60 Mousemonoclonal IgG 1:500 MilliporeMAB377 AB_2298772 NEUROD2 HumanNeuroD2 amino acids 23-37 Goat polyclonal IgG 1:500 St John’s Lab STJ72554 AB_2927398 NR1 Fusion protein containing sequence from the intracellular loop between transmembrane regions III and IV of NR1, clone 54.1 Mousemonoclonal IgG 1:100 Thermo-Fisher 32-0500 AB_2533060 PHF14 Human PHF14, 16 amino acids from theN-terminal half Rabbit polyclonal IgG 1:1000 (WB) Millipore ABE1359-25UL AB_2924888 Phospho-Histone H3 Phospho-HistoneH3 (Ser10), clone 6G3 Mousemonoclonal IgG 1:100 Cell Signaling Technology 9706 AB_331748 RAI1 Mouse Rai1 amino acids 1738-1756 Rabbit polyclonal IgG 1:200 (IF) In-houseAB_2921229 RAI1 Human RAI1 amino acids 1-100 Rabbit polyclonal IgG 1:1000 (WB) AbcamAB_1925378 RARα Recombinant Human RARα, clone 9α−9A6 Mousemonoclonal IgG 1:500 Millipore 04-1545 AB_10615821 REELIN Mouse Reelin amino acids 150-500, clone G10 Mousemonoclonal IgG 1:500 Abcam ab78540 AB_1603148 S100β Bovine brain S-100β, clone SH-B1 Mousemonoclonal IgG 1:500 Sigma S2532 AB_477499 SOX2 SRY (sex determining region Y)-box 2 Rabbit polyclonal IgG 1:100 Thermo-Fisher PA1-094AB_2539862 TBR1 T-box braintranscription factor 1 Chicken polyclonal antibodies 1:100 Millipore AB2261AB_10615497 TCF20 Human TCF20 amino acids 1909-1958 Rabbit polyclonal IgG 1:100 (IF) Millipore SAB2106444 AB_2921230 GFAP Recombinant Protein corresponding to aa1 to 432 from humanGFAP Polyclonalguinea pig antiserum 1:1000 Synaptic Systems 173004 residing near the cortical ventricles, and we found coexpression of GFAPwith a proliferating cellular marker p-HH3.

Techniques: Quantitative Proteomics, Knock-Out, Clone Assay, Generated, Western Blot, Quantitative RT-PCR, Over Expression

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma

doi: 10.1016/j.jcmgh.2018.07.003

Figure Lengend Snippet: Antibody List

Article Snippet: Mouse anti-RUNX3/AML2 (D9K6L) mAb , 13089s , WB IHC , Cell Signaling Technology.

Techniques:

Journal: Oncogene

Article Title: Threonine 209 phosphorylation on RUNX3 by Pak1 is a molecular switch for its dualistic functions.

doi: 10.1038/onc.2016.18

Figure Lengend Snippet: Figure 1. PAK1 phosphorylates Runx3 in vitro at Threonine 209 site. In vitro kinase assay with (a) GST, GST-Runx3 and myelin basic protein (positive control) as substrate and PAK1 as enzyme, (b) GST-Runx3 in presence or absence of PAK1 enzyme and (c) GST, GST-Runx3 (AA 1–415), GST-Runx3 (AA 1–57), GST-Runx3 (AA 56–187) and GST-Runx3 (AA 182–415) as substrate and PAK1 as enzyme. Pak1 was probed separately by western blotting. (d) Bioinformatic prediction of plausible PAK1 phosphorylation site in GST-Runx3 (AA 182–415) domain. (e) In vitro kinase assay with GST, GST-Runx3 (wt) and GST-Runx3 site-specific mutants—T209A, S218A and T358A—as substrate and PAK1 as enzyme. In all kinase assay images, GST protein represents ponceau-stained blot illustrating equal loading of proteins and phospho image represents autoradiogram image illustrating phosphorylated protein bands. Pak1 was probed sepa- rately by western blotting.

Article Snippet: Antibodies, reagents and chemicals The following antibodies were used: Runx3 (R3-5G4, Santa Cruz Biotechnology, Dallas, TX, USA), PAK1 (Cell Signaling Technology, Beverly, MA, USA), Vinculin (Sigma Aldrich, St Louis, MO, USA), β-Actin (Sigma Aldrich), T7 (Bethyl labs, Montgomery, TX, USA), GST (Millipore, Darmstadt, Germany), 6xHis (Millipore), Anti-PARP and anti-paxillin antibodies (Cell Signaling Technology) and anti-phosphothreonine (Millipore).

Techniques: In Vitro, Kinase Assay, Positive Control, Western Blot, Phospho-proteomics, Staining

Journal: Oncogene

Article Title: Threonine 209 phosphorylation on RUNX3 by Pak1 is a molecular switch for its dualistic functions.

doi: 10.1038/onc.2016.18

Figure Lengend Snippet: Figure 2. PAK1 phosphorylates RUNX3 in cellular context at Threonine 209. (a) Cos-1 cells were transfected with pcDNA, T7-RUNX3 (wt) or T7-RUNX3 (T209A) plasmids. After 24 h, transfected cells were metabolically labeled with p32-orthophosphoric acid overnight, followed by T7 immunoprecipitation from cell lysates and gel electrophoresis. Phosphorylated protein bands were analyzed with autoradiogram images and subsequent immunoblotting with anti-T7 antibody. (b) Cos-1 cells were transfected with pcDNA or T7-RUNX3 (wt) plasmids. After 24 h, transfected cells were serum-starved and metabolically labeled with p32-orthophosphoric acid overnight. Cells were then treated with EGF (100 ng/ml), Sphingosine (100 μM) or 10% fetal bovine serum for 30 min, followed by T7 immunoprecipitation from cell lysates and gel electrophoresis. Phosphorylated protein bands were analyzed with autoradiogram images and subsequent immunoblotting with anti-T7 antibody. (c) Cos-1 cells were co-transfected with pcDNA or T7-RUNX3 (wt) plasmids and control or PAK1 siRNA. After 24 h, transfected cells were metabolically labeled with p32-orthophosphoric acid overnight, followed by T7 immunoprecipitation from cell lysates and gel electrophoresis. Phosphorylated protein bands were analyzed with autoradiogram images and subsequent immunoblotting with anti-T7 antibody. PAK1 knockdown efficacy was determined by immunoblotting with anti-PAK1 antibody with actin as a house-keeping gene. (d) Cos-1 cells were transfected with pcDNA or T7-RUNX3 (wt) plasmids. After 24 h, transfected cells were serum-starved and metabolically labeled with p32-orthophosphoric acid overnight. Cells were then treated with 20 μM IPA-3 (PAK1 inhibitor) or 20 μM PIR-3.5 (structural analog of IPA-3) for 30 min, followed by T7 immunoprecipitation from cell lysates and gel electrophoresis. Phosphorylated protein bands were analyzed with autoradiogram images and subsequent immunoblotting with anti-T7 antibody.

Article Snippet: Antibodies, reagents and chemicals The following antibodies were used: Runx3 (R3-5G4, Santa Cruz Biotechnology, Dallas, TX, USA), PAK1 (Cell Signaling Technology, Beverly, MA, USA), Vinculin (Sigma Aldrich, St Louis, MO, USA), β-Actin (Sigma Aldrich), T7 (Bethyl labs, Montgomery, TX, USA), GST (Millipore, Darmstadt, Germany), 6xHis (Millipore), Anti-PARP and anti-paxillin antibodies (Cell Signaling Technology) and anti-phosphothreonine (Millipore).

Techniques: Transfection, Metabolic Labelling, Labeling, Immunoprecipitation, Nucleic Acid Electrophoresis, Western Blot, Control, Knockdown

Journal: Oncogene

Article Title: Threonine 209 phosphorylation on RUNX3 by Pak1 is a molecular switch for its dualistic functions.

doi: 10.1038/onc.2016.18

Figure Lengend Snippet: Figure 3. PAK1 physically interacts with RUNX3. (a) Characterization of anti-phospho-RUNX3 (T209) antibody using in vitro PAK1 kinase assay; comparing GST-Runx3 phosphorylation in the presence or absence of PAK1. Lower panel illustrates hot kinase assay (with P32- labeled ATP) and upper panel includes parallel cold reaction immunoblotted with anti-phospho-Runx3 (T209) antibody. (b) In vitro kinase assay with GST, GST-Runx3 and GST-Runx3 (T209A) as substrate in the presence or absence PAK1 enzyme was performed. Proteins were immunoblotted with anti-phospho-Runx3 (T209) antibody. (c) Cos-1 cells were transfected with pcDNA-Flag, Flag-RUNX3 or Flag-Runx3 (T209A). Total cell lysates were resolved on SDS–PAGE and immunoblotting is performed for anti-phospho-Runx3 (T209), anti-flag and anti-vinculin (house-keeping gene) antibodies. (d) Total cell lysates from SCC-131 stable PAK1 knockdown clone and non-target clone along with transient T7-Runx3- transfected Cos-1 cells (as positive control), were resolved by electrophoresis and immunoblotted for anti-phospho-Runx3 (T209), anti-Runx3, anti-PAK1 and anti-vinculin (house-keeping gene) antibodies. (e) Pak1 overexpression induces Runx3 phosphorylation at Threonine 209. Cos-1 cells were transfected with indicated concentration of Pak1 and Flag Runx3 and immunoblotted for anti-phospho-Runx3 (T209). (f) GST pull-down assay was performed with GST-Runx3 as a bait and T7-PAK1 as a prey (Left panel) and GST-PAK1 as a bait and T7-Runx3 as a prey (right panel). (g) Representative sensorgram showing the binding of GST-Runx3 with the surface-immobilized PAK1. The red and blue curves represent the binding curves for 1000 nM and 100 nM concentrations of GST-Runx3, respectively. The surface immobilization of PAK1 was upto 3000 RUs. The KD value of 4.38 × 10 −8 M was determined for Pak1-Runx3 interaction from the above two concentrations using Langmuir kinetic model. (h) Co-immunoprecipitation analysis was performed from SCC-131 total cell lysate by immunoprecipitating PAK1 and immunoblotting RUNX3 (left panel) and immunoprecipitating RUNX3 and immunoblotting PAK1 (right panel). In all kinase assay images, GST protein represents ponceau-stained blot illustrating equal loading of proteins and phospho image represents autoradiogram image illustrating phosphorylated protein bands.

Article Snippet: Antibodies, reagents and chemicals The following antibodies were used: Runx3 (R3-5G4, Santa Cruz Biotechnology, Dallas, TX, USA), PAK1 (Cell Signaling Technology, Beverly, MA, USA), Vinculin (Sigma Aldrich, St Louis, MO, USA), β-Actin (Sigma Aldrich), T7 (Bethyl labs, Montgomery, TX, USA), GST (Millipore, Darmstadt, Germany), 6xHis (Millipore), Anti-PARP and anti-paxillin antibodies (Cell Signaling Technology) and anti-phosphothreonine (Millipore).

Techniques: In Vitro, Kinase Assay, Phospho-proteomics, Labeling, Transfection, SDS Page, Western Blot, Knockdown, Positive Control, Electrophoresis, Over Expression, Concentration Assay, Pull Down Assay, Binding Assay, Immunoprecipitation, Staining

Journal: Oncogene

Article Title: Threonine 209 phosphorylation on RUNX3 by Pak1 is a molecular switch for its dualistic functions.

doi: 10.1038/onc.2016.18

Figure Lengend Snippet: Figure 4. PAK1 phosphorylation sequesters RUNX3 in cytoplasmic compartment. (a) Immunofluorescence staining was performed on SCC-131 stable PAK1 knockdown and non-target control cells against RUNX3 (Alexa 488), PAK1 (Alexa 546) and DAPI (nuclear staining). (b) Subcellular fractions were isolated from SCC-131 stable PAK1 knockdown and non-target control cells, and separated using gel electrophoresis. Following the same, immunoblotting was performed with anti-RUNX3, anti-PAK1, anti-Paxillin (cytoplasmic marker) and anti-PARP (nuclear marker).

Article Snippet: Antibodies, reagents and chemicals The following antibodies were used: Runx3 (R3-5G4, Santa Cruz Biotechnology, Dallas, TX, USA), PAK1 (Cell Signaling Technology, Beverly, MA, USA), Vinculin (Sigma Aldrich, St Louis, MO, USA), β-Actin (Sigma Aldrich), T7 (Bethyl labs, Montgomery, TX, USA), GST (Millipore, Darmstadt, Germany), 6xHis (Millipore), Anti-PARP and anti-paxillin antibodies (Cell Signaling Technology) and anti-phosphothreonine (Millipore).

Techniques: Phospho-proteomics, Staining, Knockdown, Control, Isolation, Nucleic Acid Electrophoresis, Western Blot, Marker

Journal: Oncogene

Article Title: Threonine 209 phosphorylation on RUNX3 by Pak1 is a molecular switch for its dualistic functions.

doi: 10.1038/onc.2016.18

Figure Lengend Snippet: Figure 5. Differential localization of RUNX3 constructs. Cos-1 cells were transiently transfected with mammalian expression plasmid pcDNA-6A, tagged with FLAG. Cells were seeded on coverslips 24 h post transfection and cultured for another 24 h. Cells were probed using anti-FLAG antibody and anti-Pak1 antibody. Green coupled (Alexa 488) anti-mouse and red coupled (Alexa 546) anti-rabbit antibodies were used to visualize FLAG-tagged RUNX3 and PAK1, respectively. Wild-type cells showed predominant localization of RUNX3 in the cytoplasm, whereas T209A showed nuclear localization. T209E phospho-mimic cells showed cytoplasmic localization of RUNX3 similar to the wild type. All cells showed the presence of endogenous Pak1, indicating that the localization of Wt-RUNX3 is altered because of phosphorylation by Pak1.

Article Snippet: Antibodies, reagents and chemicals The following antibodies were used: Runx3 (R3-5G4, Santa Cruz Biotechnology, Dallas, TX, USA), PAK1 (Cell Signaling Technology, Beverly, MA, USA), Vinculin (Sigma Aldrich, St Louis, MO, USA), β-Actin (Sigma Aldrich), T7 (Bethyl labs, Montgomery, TX, USA), GST (Millipore, Darmstadt, Germany), 6xHis (Millipore), Anti-PARP and anti-paxillin antibodies (Cell Signaling Technology) and anti-phosphothreonine (Millipore).

Techniques: Construct, Transfection, Expressing, Plasmid Preparation, Cell Culture, Phospho-proteomics

Journal: Oncogene

Article Title: Threonine 209 phosphorylation on RUNX3 by Pak1 is a molecular switch for its dualistic functions.

doi: 10.1038/onc.2016.18

Figure Lengend Snippet: Figure 6. PAK1 phosphorylation switches off the tumor suppressor activity of RUNX3. (a) Cos-1 cells were co-transfected with p21CIP1/WAF1 promoter luciferase reporter construct and pcDNA or flag-RUNX3 (wt) or flag-RUNX3 (T209A) plasmid. After 24 h, cells were lysed; luciferase activity was measured (n = 3) and normalized with respective β-galactosidase activity. Each value represents mean ± s.e.m. **Po0.001 and *Po0.05, as compared with respective value. (b) Simultaneously, total cell lysates from (a) were separated using electrophoresis and were analyzed with immunoblotting using anti-p21, anti-flag, anti-Cyclin D1 and anti-vinculin (house-keeping) antibodies. Cyclin D1 and corresponding vinculin were run separately on another gel. (c) Cos-1 cells were co-transfected with p21CIP/WAF-1 promoter luciferase reporter construct and pCMV or flag-RUNX3 alone or flag-RUNX3 along with PAK1 (T423E) or flag-RUNX3 along with PAK1 (K299R). After 24 h, cells were lysed; luciferase activity was measured (n = 3) and normalized with respective β-galactosidase activity. Each value represents mean ± s.e.m. **Po0.0029, *Po0.05 and ns: non significant, as compared with respective value.

Article Snippet: Antibodies, reagents and chemicals The following antibodies were used: Runx3 (R3-5G4, Santa Cruz Biotechnology, Dallas, TX, USA), PAK1 (Cell Signaling Technology, Beverly, MA, USA), Vinculin (Sigma Aldrich, St Louis, MO, USA), β-Actin (Sigma Aldrich), T7 (Bethyl labs, Montgomery, TX, USA), GST (Millipore, Darmstadt, Germany), 6xHis (Millipore), Anti-PARP and anti-paxillin antibodies (Cell Signaling Technology) and anti-phosphothreonine (Millipore).

Techniques: Phospho-proteomics, Activity Assay, Transfection, Luciferase, Construct, Plasmid Preparation, Electrophoresis, Western Blot

Journal: Oncogene

Article Title: Threonine 209 phosphorylation on RUNX3 by Pak1 is a molecular switch for its dualistic functions.

doi: 10.1038/onc.2016.18

Figure Lengend Snippet: Figure 7. T209 phosphorylation on Runx3 by Pak1 is a switch regulating its dualistic functions. (a) Panc-28 stable overexpressing clones transfected with pcDNA, Wt-Runx3, Runx3-T209A and Runx3-T209E; 100 ug of total protein lysates were used for Runx3 probing. (b and c) Anchorage- independent soft agar and Clonogenic cell survival assay. Panc-28 cells stably overexpressing Wt-Runx3 and Runx3-T209A showed a significant decrease in the number of colonies formed in both the assays, whereas stably overexpressing Runx3-T209E Panc-28 cells showed a significant increase in the number of colonies when compared with vector control (pcDNA) cells. Each value represents the mean ±s.e.m. ***Po0.001, **Po0.005 compared with vector clones. (d) Representative tumor xenografts images of Panc-28 stable overexpressing clones of pcDNA, Wt-Runx3, Runx3-T209A and Runx3-T209E implanted subcutaneously in nude mice. (e) Graph showing tumor growth in nude mouse xenografts.

Article Snippet: Antibodies, reagents and chemicals The following antibodies were used: Runx3 (R3-5G4, Santa Cruz Biotechnology, Dallas, TX, USA), PAK1 (Cell Signaling Technology, Beverly, MA, USA), Vinculin (Sigma Aldrich, St Louis, MO, USA), β-Actin (Sigma Aldrich), T7 (Bethyl labs, Montgomery, TX, USA), GST (Millipore, Darmstadt, Germany), 6xHis (Millipore), Anti-PARP and anti-paxillin antibodies (Cell Signaling Technology) and anti-phosphothreonine (Millipore).

Techniques: Phospho-proteomics, Clone Assay, Transfection, Clonogenic Cell Survival Assay, Stable Transfection, Plasmid Preparation, Control

Journal: Oncology reports

Article Title: Unfavorable clinical implications for hypermethylation of RUNX3 in patients with salivary gland adenoid cystic carcinoma.

doi: 10.3892/or.2011.1282

Figure Lengend Snippet: Figure 1. Real-time RT-PCR analysis of RUNX3 mRNA in ACC-2, ACC-M and ACC-3 cells with or without 5-Aza-dC treatment (M, DNA marker DL2000). (A) Electrophoresis of RUNX3 mRNA in ACC-2, ACC-M and ACC-3 without 5-Aza-dC treatment. (B) Electrophoresis of β‑actin mRNA in ACC-2, ACC-M and ACC-3 without 5-Aza-dC treatment. (C) Electrophoresis of RUNX3 mRNA in ACC-2, ACC-M and ACC-3 after 300 nmol/l 5-Aza-dC treatment for 72 h. (D) Electrophoresis of β‑actin mRNA in ACC-2, ACC-M and ACC-3 after 300 nmol/l 5-Aza-dC treatment for 72 h.

Article Snippet: Goat anti-human RUNX3 polyclonal antibody (diluted x30; R&D Systems Inc., USA) was added and incubation was carried out for 1 h at 37 ̊C.

Techniques: Quantitative RT-PCR, Marker, Electrophoresis

Journal: Oncology reports

Article Title: Unfavorable clinical implications for hypermethylation of RUNX3 in patients with salivary gland adenoid cystic carcinoma.

doi: 10.3892/or.2011.1282

Figure Lengend Snippet: Figure 2. LSCM results of RUNX3 protein in ACC-2, ACC-M and ACC-3 cells with or without 5-Aza-dC treatment. (A) ACC-2 cells without 5-Aza-dC treatment. (B) ACC-2 cells treated with 300 nmol/l 5-Aza-dC for 72 h. (C) ACC-M cells without 5-Aza-dC treatment. (D) ACC-M cells treated with 300 nmol/l 5-Aza-dC for 72 h. (E) ACC-3 cells without 5-Aza-dC treatment. (F) ACC-3 cells treated with 300 nmol/l 5-Aza-dC for 72 h.

Article Snippet: Goat anti-human RUNX3 polyclonal antibody (diluted x30; R&D Systems Inc., USA) was added and incubation was carried out for 1 h at 37 ̊C.

Techniques:

Journal: Oncology reports

Article Title: Unfavorable clinical implications for hypermethylation of RUNX3 in patients with salivary gland adenoid cystic carcinoma.

doi: 10.3892/or.2011.1282

Figure Lengend Snippet: Figure 3. Western blotting results of RUNX3 in ACC-2, ACC-M and ACC-3 cells with or without 5-Aza-dC treatment. R/G represents the brightness ratio of the RUNX3 protein (45 kDa) vs. GAPDH (36 kDa).

Article Snippet: Goat anti-human RUNX3 polyclonal antibody (diluted x30; R&D Systems Inc., USA) was added and incubation was carried out for 1 h at 37 ̊C.

Techniques: Western Blot

Journal: Oncology reports

Article Title: Unfavorable clinical implications for hypermethylation of RUNX3 in patients with salivary gland adenoid cystic carcinoma.

doi: 10.3892/or.2011.1282

Figure Lengend Snippet: Figure 4. The relative expression of RUNX3 protein in primary salivary gland adenoid cystic carcinoma compared to that in normal salivary gland adenoid cystic tissue (﹡P<0.001).

Article Snippet: Goat anti-human RUNX3 polyclonal antibody (diluted x30; R&D Systems Inc., USA) was added and incubation was carried out for 1 h at 37 ̊C.

Techniques: Expressing

Journal: Oncology reports

Article Title: Unfavorable clinical implications for hypermethylation of RUNX3 in patients with salivary gland adenoid cystic carcinoma.

doi: 10.3892/or.2011.1282

Figure Lengend Snippet: Figure 5. Comparison of disease-free survival curves for patients with RUNX3 promoter methylation.

Article Snippet: Goat anti-human RUNX3 polyclonal antibody (diluted x30; R&D Systems Inc., USA) was added and incubation was carried out for 1 h at 37 ̊C.

Techniques: Comparison, Methylation